欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2009, Vol. 35 ›› Issue (7): 1202-1208.doi: 10.3724/SP.J.1006.2009.01202

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝自交不亲和信号传导元件ARC1的体外表达及其与SRK相互作用验证

牛义1,王志敏1,高启国1,宋明1,王小佳1,*,朱利泉2,*   

  1. 1西南大学重庆市蔬菜重点实验室；2西南大学植物生理生物化学实验室，重庆400716
  • 收稿日期:2008-12-05 修回日期:2009-03-16 出版日期:2009-07-12 网络出版日期:2009-05-18
  • 通讯作者: 王小佳,E-mail:wxj@swu.edu.cn;朱利泉,E-mail:zhuliquan@swu.edu.cn;Tel:023-68250794;Fax:023-68251914
  • 基金资助:

    本研究由国家自然科学基金项目（30471190和30671429），重庆市自然科学基金项目（9266），高校博士点基金项目（20050625003），西南大学科研基金项目（SWU208044）资助。

Expression of ARC1 in Vitro and Test of Interaction between ARC1 and SRK from Brassica oleracea L. in Signal Transduction Pathway of Self-Incompatibility

NIU Yi1,WANG Zhi-Min1,GAO Qi-Guo1,SONG Ming1,WANG Xiao-Jia1*,ZHU Li-Quan2*   

  1. 1Key Laboratory in Olericulture of Chongqing，Southwest University，2Plant Physiology and Biochemistry Laboratory of Southwest University，Chongqing 400716，China
  • Received:2008-12-05 Revised:2009-03-16 Published:2009-07-12 Published online:2009-05-18
  • Contact: WANG Xiao-Jia,E-mail:wxj@swu.edu.cn;ZHU Li-Quan,E-mail:zhuliquan@swu.edu.cn;Tel:023-68250794;Fax: 023-68251914

PDF

1326

被引次数

5

摘要：

在甘蓝自交不亲和信号传导中ARC1和上游因子SRK之间可能存在相互作用。为进一步证实该相互作用，以甘蓝E1为材料，采用RT-PCR技术扩增ARC1的编码序列, 构建ARC1原核表达质粒pET43.1a-ARC1，转化宿主菌大肠杆菌BL21，通过SDS-PAGE检测该蛋白的表达。利用免疫共沉淀原理及pET43.1a-ARC1融合蛋白序列中的6×His标签与Ni+结合的特点建立了体外检测蛋白质相互作用的新方法, 并用该方法对ARC1与SRK的相互作用进行了检测。结果表明，在体外ARC1能与SRK相互作用并形成复合体，这为深入分析ARC1与SRK相互作用机理以及探讨ARC1与下游传导元件的相互作用提供了理论和技术基础。

关键词: 甘蓝, 信号传导, S-位点体激酶（SRK）, 臂重复蛋白1（ARC1）

Abstract:

Self-incompatibility (SI) is a widespread mechanism in flowering plants that prevents inbreeding and promotes outcrossing. Self-pollen recognition relies on the products of genes located at the S (self-incompatibility) locus. Significant progress has been made in understanding molecular interactions allowing stigmatic cells to recognize and reject self-pollen in Brassica during the past decades. Thus, the male and female determinants responsible for the self-incompatibility (SI) response have been identified. The structural features of these molecules strongly suggest that SI response is triggered by a ligand-receptor interaction. ARC1 is anArm Repeat Containing and also a downstream gene of SRK. ARC1 interacts with SRK probably in signal transduction pathway of self-incompatibility. In this study, with an aim to testify the interaction, the coding sequences of ARC1 were amplified from stigma mRNA of Brassica oleracea L., and inserted into the expression vector pET43.1a to construct the recombinant plasma pET43.1a-ARC1, transformed E. coli(BL21) and detected expression of the recombinant protein via SDS-PAGE. Using Co-Immunoprecipitation theory and characteristic of 6×His Tag combined with Ni+ in pET43.1a-ARC1, a new method was put forward for testing the interaction between proteins. Through the method the interaction between ARC1 and SRKwas analyzed, showing that ARC1 and SRK could act with each other to combine and form a complex. This research provides a theoretical and technical bases for further analyzing the mechanism of interaction between ARC1 and SRK, for probing into interaction between ARC1 and downstream targets.

Key words: Brassica oleracea L, Signal transduction, S-locus receptor Kinase（SRK）, Arm repeat containing1（ARC1）

[1] McCubbin A G, Kao T H. Molecular recognition and response in pollen and pistil interactions. Annu Rev Cell Dev Biol 2000, 16: 333-364
[2] Nasrallah J B, Nasrallah M E. Pollen-stigma signaling in the sporophytic self-incompatibility response. Plant Cell, 1993, 5: 1325-1335
[3] Conner J A, Conner P, Nasrallah J B. Comparative mapping of the Brassica S locus region and its homology in Arabidopsis: implications for the evolution of mating systems in the Brassicaceae. Plant Cell, 1998, 10: 801-812
[4] Nasrallah J B, Kao T H, Goldberg M L, Nasrallah M E. A cDNA clone encoding an s-locus specific glycoprotein from Brassica oleracea L. Nature, 1985, 318: 263-267

[5] Watanabe M, Ono T, Hatakeyama K. Molecular characterization of SLG and S-related genes in a self-incompatible Brassica campestris L. var. yellow sarson. Sex Plant Reprod, 1997, 10: 332-340
[6] Kusaba M, Nishio T, Satta Y, Hinata K, Ockendon D. Striking sequence similarity in inter-and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris: Implications for the evolution and recognition mechanism. Proc Natl Acad Sci USA, 1997, 94: 7673-7678
[7] Hatakeyama K, Takasaki T, Watanabe M, Hinata K. Molecular characterization of S locus genes, SLG and SRK, in a pollen-recessive self-incompatibility haplotype of Brassica rapa L. Genetics,1998, 149: 1587-1597
[8] Doughty J, Dixon S, Hiscock S J, Willis A C, Parkin I A P, Dickinson H G.PCP-A1, a defensin-like Brassica pollen coat protein that binds the S locus glycoprotein, is the product of gametophytic gene expression. Plant Cell, 1998, 10: 1333-1348
[9] Schopfer C R, Nasrallah M E, Nasrallah J B. The male determinant of self-incompatibility in Brassica. Science, 1999, 286: 1697-1700
[10] Suzuki G, Kai N, Hisrose T, Fukui K, Nishio T, Takayama S, Isogai A, Watanabe M, Hinata K. Genomic organization of the S-locus: Identification and characterization of genes in the SLG/SRK region of S9 haplotype of Brassica campestris (syn. rapa). Genetics, 1999, 153: 391-400
[11] Takayama S, Shiba H, Iwano M, Shimosato H, Che F S, Kai N, Watanabe M, Suzuki G, Hinata K, Isogai A. The pollen determinant of self-incompatibility in Brassica campestris. Proc Natl Acad Sci USA, 2000, 97: 1920-1925
[12] Stein J C, Howlett B, Boyes D C, Nasarllah M E, Nasarllah J B. Molecular cloning of a putative receptor protein kinas encoded at the self-incompatibility locus of Brassica oleracea.Proc Natl Acad Sci USA, 1991, 88: 8816-8820

[13] Stein J C, Nasrallah J B. A plant receptor-like gene, the S-locus receptor kinase of Brassica oleracea, encodes a functional serine/threonine kinase. Plant Physiol, 1993, 101: 1103-1106
[14] Giranton J L, Duma C, Cock M, Gaude T. The integral membrane S-locus receptor kinase of Brassica has serine/threonine kinase activity in a membranous environment and spontaneously forms oligomers in planta. Proc Natl Acad Sci USA, 2000, 97: 3759-3764


[15] Shiu S H, Bleecker A B. Plant receptor-like kinase gene family: Diversity, function and signaling. Sci STKE, 2001, 113: RE22


[16] Stein J C, Dixit R, Nasrallah M E, Nasrallah J B. SRK, the stigma specific S-locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco. Plant Cell, 1996, 8: 429-445

[17] Cabrillac D, Cock J M, Dumas C. The S-locus receptor kinase is inhibited by thioredoxins and activated by pollen coat proteins. Nature, 2001, 410: 220-223

[18] Goring D R, Rothstein S J. The S-locus receptor kinase gene in a self-Incompatible Brassica napus line encodes a functional serine/threonine kinase. Plant Cell, 1992, 4: 1273-1281

[19] Takasaki T, Hatakeyama K, Suzuki G, Watanabe M, Isogaill A, Hinata K. The S receptor kinase determines self-incompatibility in Brassica stigma. Nature, 2000, 403: 913-916
[20] Cui Y, Bi Y M, Brugiere N, Amoldo M, Rothstein S J. The S locus glycoprotein and the S receptor kinase are sufficient for self-pollen rejection in Brassica. Proc Natl Acad Sci USA, 2000, 97: 3713-3717
[21] Silva, N F, Goring D R. Mechanisms of self-incompatibility in flowering plants. Cell Mol Life Sci, 2001, 58: 1988-2007
[22] Silva N F, Stone S L, Christe L N, Sulaman W, Nazarain K A P, Burnett M, Arnoldo M A, Rothstein S J, Goring D R. Expression of the S receptor kinase in self-compatible Brassica napus cv. Westar leads to the allele-specific rejection of self-incompatible Brassica napus pollen. Mol Gen Genet, 2001, 265: 552-559
[23] Gu T, Mazzurco M, Sulaman W, Matias D, Goring D R. Binding of an arm repeat protein to the kinase domain of the S-locus receptor kinase. Proc Natl Acad Sci USA, 1998, 95: 382-387
[24] Stone S L, Arnoldo M A, Goring D R.A breakdown of Brassica self-incompatibility in ARC1 antisense transgenic plants. Science, 1999, 286: 1729-1731
[25] Mazzurco M, Sulaman W, Elina H, Cock J M, D R Goring. Further analysis of the interactions between the Brassica S receptor kinase and three interacting proteins (ARCl, THLl and THL2) in the yeast two-hybrid system. Plant Mol Biol, 2001, 45: 365-376
[26] Liu D(刘东), Zhu L-Q(朱利泉), Wang X-J(王小佳). Cloning and characterization of encoding sequence of srk-binding protein ARC1 from Brassica olereacea L in self-incompatibility signaling process. Acta Hort Sin (园艺学报), 2004, 30: 427-431(in Chinese with English abstract)
[27] Gao Q-G(高启国), Song M(宋明), Niu Y(牛义), Yang K(杨昆), Zhu L-Q(朱利泉), Wang X-J(王小佳). Test of interaction between THL1 and SRK from Brassica oleracea L. in self-incompatibility signaling process. Acta Agron Sin (作物学报), 2008, 34(6): 1-10(in Chinese with English abstract)

[28] He F-C(贺福初), Qian X-H(钱小红), Zhang X-M(张学敏). Protein-Protein Interactions (蛋白质-蛋白质相互作用), 2nd edn. Beijing: China Agriculture Press, 2004. pp 47-56(in Chinese)
[29] Sobotka R, Sakova L, Curn V. Molecular Mechanisms of Self-Incompatibility in Brassica. Mol Biol, 2000, 2: 103-112
[1] 陈松余, 丁一娟, 孙峻溟, 黄登文, 杨楠, 代雨涵, 万华方, 钱伟. 甘蓝型油菜BnCNGC基因家族鉴定及其在核盘菌侵染和PEG处理下的表达特性分析[J]. 作物学报, 2022, 48(6): 1357-1371.
[2] 秦璐, 韩配配, 常海滨, 顾炽明, 黄威, 李银水, 廖祥生, 谢立华, 廖星. 甘蓝型油菜耐低氮种质筛选及绿肥应用潜力评价[J]. 作物学报, 2022, 48(6): 1488-1501.
[3] 张以忠, 曾文艺, 邓琳琼, 张贺翠, 刘倩莹, 左同鸿, 谢琴琴, 胡燈科, 袁崇墨, 廉小平, 朱利泉. 甘蓝S-位点基因SRK、SLG和SP11/SCR密码子偏好性分析[J]. 作物学报, 2022, 48(5): 1152-1168.
[4] 袁大双, 邓琬玉, 王珍, 彭茜, 张晓莉, 姚梦楠, 缪文杰, 朱冬鸣, 李加纳, 梁颖. 甘蓝型油菜BnMAPK2基因的克隆及功能分析[J]. 作物学报, 2022, 48(4): 840-850.
[5] 黄成, 梁晓梅, 戴成, 文静, 易斌, 涂金星, 沈金雄, 傅廷栋, 马朝芝. 甘蓝型油菜BnAPs基因家族成员全基因组鉴定及分析[J]. 作物学报, 2022, 48(3): 597-607.
[6] 王瑞, 陈雪, 郭青青, 周蓉, 陈蕾, 李加纳. 甘蓝型油菜白花基因InDel连锁标记开发[J]. 作物学报, 2022, 48(3): 759-769.
[7] 谢琴琴, 左同鸿, 胡燈科, 刘倩莹, 张以忠, 张贺翠, 曾文艺, 袁崇墨, 朱利泉. 甘蓝自交不亲和相关基因BoPUB9的克隆及表达分析[J]. 作物学报, 2022, 48(1): 108-120.
[8] 王艳花, 刘景森, 李加纳. 整合GWAS和WGCNA筛选鉴定甘蓝型油菜生物产量候选基因[J]. 作物学报, 2021, 47(8): 1491-1510.
[9] 左香君, 房朋朋, 李加纳, 钱伟, 梅家琴. 有毛野生甘蓝(Brassica incana)抗蚜虫特性研究[J]. 作物学报, 2021, 47(6): 1109-1113.
[10] 李杰华, 端群, 史明涛, 吴潞梅, 柳寒, 林拥军, 吴高兵, 范楚川, 周永明. 新型抗广谱性除草剂草甘膦转基因油菜的创制及其鉴定[J]. 作物学报, 2021, 47(5): 789-798.
[11] 唐鑫, 李圆圆, 陆俊杏, 张涛. 甘蓝型油菜温敏细胞核雄性不育系160S花药败育的形态学特征和细胞学研究[J]. 作物学报, 2021, 47(5): 983-990.
[12] 周新桐, 郭青青, 陈雪, 李加纳, 王瑞. GBS高密度遗传连锁图谱定位甘蓝型油菜粉色花性状[J]. 作物学报, 2021, 47(4): 587-598.
[13] 李书宇, 黄杨, 熊洁, 丁戈, 陈伦林, 宋来强. 甘蓝型油菜早熟性状QTL定位及候选基因筛选[J]. 作物学报, 2021, 47(4): 626-637.
[14] 张春, 赵小珍, 庞承珂, 彭门路, 王晓东, 陈锋, 张维, 陈松, 彭琦, 易斌, 孙程明, 张洁夫, 傅廷栋. 甘蓝型油菜千粒重全基因组关联分析[J]. 作物学报, 2021, 47(4): 650-659.
[15] 唐婧泉, 王南, 高界, 刘婷婷, 文静, 易斌, 涂金星, 傅廷栋, 沈金雄. 甘蓝型油菜SnRK基因家族生物信息学分析及其与种子含油量的关系[J]. 作物学报, 2021, 47(3): 416-426.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2